CN106967202B

CN106967202B - Nano-gel enzyme and preparation and application thereof

Info

Publication number: CN106967202B
Application number: CN201710126897.6A
Authority: CN
Inventors: 陈彦涛; 陈正鹏子; 郑桂钦; 龙玲
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2017-03-06
Filing date: 2017-03-06
Publication date: 2020-05-22
Anticipated expiration: 2037-03-06
Also published as: CN106967202A

Abstract

The invention discloses a nano-gel enzyme and preparation and application thereof. The preparation method of the nanogel enzyme comprises the following steps: (1) chemically modifying horseradish peroxidase by using N-acryloyloxy succinimide to prepare a horseradish peroxidase-N-acryloyloxy succinimide macromonomer; (2) and (2) carrying out free radical polymerization reaction on a horseradish peroxidase-N-acryloyloxy succinimide macromonomer and an alkene monomer with zwitterion characteristics to prepare the nano-gel enzyme. The preparation method of the nano-gel enzyme is green and environment-friendly, mild in reaction condition, low in cost and easy to industrialize. The nano-gel enzyme has good water solubility, hydrophilicity and biocompatibility, can effectively remove dyes, and can be directly used as a degradation material of dye industrial wastewater.

Description

Nano-gel enzyme and preparation and application thereof

Technical Field

The invention relates to the technical field of nano enzyme, in particular to a horseradish peroxidase nano gel enzyme and preparation and application thereof.

Background

With printing and dyeingDue to the development of the material industry, dye wastewater becomes a current main water pollution source and has great harm to the environment and organisms. However, 12% of industrial wastewater is not treated at all, and the treated wastewater yield is only 63%. The printing and dyeing industry is the traditional prop industry in China, and according to incomplete statistics, the daily dye wastewater discharge amount of the China reaches 300 ten thousand meters³500 km of³The main sources of the wastewater are textile factories and printing and dyeing packaging industries.

The dye wastewater has two characteristics. Firstly, the organic matter content is high; dyes are mostly azo compounds, which pose a great threat to the health of living beings. Secondly, the azo compound has extremely high chemical stability, belongs to typical organic pollutants with high toxicity and difficult degradation, has high durability and diffusion capacity of related components, can be continuously retained in natural environments such as water, soil and the like, can cause biological enrichment, and contains carcinogenic factors. The wastewater containing azo dyes is one of the major solutions of harmful wastewater in the water pollution control in China.

Horseradish Peroxidase (HRP), also known as Peroxidase. The biological enzyme is extracted from microorganisms or plants, has high-efficiency catalytic capability, can promote a system to release a large amount of oxygen free radicals, can be regarded as an advanced form of an oxidation method, and has a plurality of advantages of the biological method. Peroxidases have attracted considerable attention in the field of environmental water treatment in recent years and are considered to be the most promising biocatalysts (Kumar, V., et al. A. Horseradirachnoid immobilized polypeptide matrix: a biocatalytic system for dye water treatment. RSC Advances,2016,6, 2976). However, the natural peroxidase has poor stability, is easy to inactivate in the catalytic process and is not suitable for recycling, and the application of the peroxidase in the catalytic field is greatly limited.

Disclosure of Invention

The invention provides a nano-gelase, which is prepared by introducing double bonds and vinyl monomers with zwitterion characteristics to carry out molecular level gelification and wrapping on horseradish peroxidase (HRP), so that the environmental adaptability is improved, the adsorption of degradation products is effectively resisted, the nano-gelase is favorable for recycling, and the aim of efficiently and environmentally degrading dye components is fulfilled by utilizing the catalytic oxidation capacity of the nano-gelase.

The invention uses commercial HRP as initial raw material. HRP is a plant peroxidase, is hemoprotein formed by a single peptide chain and prosthetic group porphyrin, has the molecular weight of 44000Da, is wide in existence and relatively low in price, and has the characteristics of high-efficiency catalytic performance and mild catalytic action, so that people find that HRP can treat various compounds containing hydroxyl groups in wastewater, such as chlorophenol, and has a good effect of catalytically degrading industrial dye wastewater, such as carbaryl dye, in the middle of 80 years.

The nanometer gel enzyme is prepared by using HRP as a raw material to carry out chemical modification, introducing double bonds and then carrying out free radical copolymerization with alkene monomers with zwitterion characteristics. The specific technical scheme is as follows:

a preparation method of nanogel enzyme comprises the following steps:

(1) chemically modifying horseradish peroxidase by using N-acryloyloxy succinimide (NAS) to prepare a horseradish peroxidase-N-acryloyloxy succinimide (HRP-NAS) macromonomer; the HRP-NAS macromonomer is a compound containing a double bond;

(2) and (2) carrying out free radical polymerization reaction on the HRP-NAS macromonomer obtained in the step (1) and a vinyl monomer with zwitterion characteristic to prepare the nano-gel enzyme.

In the step (1), the reaction conditions for chemically modifying horseradish peroxidase by using N-acryloyloxy succinimide comprise: the reaction is stirred for at least 2 hours in the solution state, so that the reaction is facilitated. In order to achieve better effects of the invention, it is preferable that: dissolving HRP in a phosphoric acid buffer solution (PBS) to obtain an HRP solution; dissolving NAS in dimethyl sulfoxide (DMSO) to obtain a NAS solution; the HRP solution was stirred and the NAS solution was added dropwise to the HRP solution and the reaction was stirred for at least 2 h. The NAS solution is preferably continuously added in one time, so that the reaction is more favorably carried out. The reaction temperature of the reaction is not strictly limited, and the reaction temperature is generally natural environment temperature, such as natural environment temperature of 18 ℃ to 35 ℃ at normal temperature or room temperature.

The phosphoric acid buffer solution can be prepared by the existing method or can be prepared by the commercial products, such as: 0.1mol/L-0.2mol/L phosphoric acid buffer solution.

The dosage of the NAS is as follows: the molar quantity of NAS is larger than or equal to that of amino groups in HRP, and the chemical modification of all amino groups in HRP can be completed by the same amount or excessive amount of NAS. Preferably: the molar quantity of NAS is 1-20 times that of the amino in HRP. More preferably: the molar quantity of NAS is 3-8 times that of the amino in HRP.

Preferably, in the step (2), the monomer terminal of the vinyl monomer having zwitterionic character has both positive and negative zwitterionic functional groups. Further preferably, the vinyl monomer having zwitterionic characteristic is one or two of a compound containing an acrylamide group and a zwitterionic functional group having a positive charge and a negative charge at the monomer end, and a compound containing an acrylate group and a zwitterionic functional group having a positive charge and a negative charge at the monomer end. For example, one or more of carboxylic acid betaine acrylamide (CBAA), carboxylic acid betaine methacrylate (CBMA), sulfobetaine methacrylate (SBMA), and 2-hydroxyethyl Methacrylate Phosphatidylcholine (MPC) can be used.

CBAA has the structural formula shown in formula I:

CBMA structural formula is shown in formula II:

the SBMA structural formula is shown in formula III:

MPC has the formula IV:

the free radical polymerization reaction can also be added with two raw materials of an initiator and a stabilizer or three raw materials of the initiator, the stabilizer and a crosslinking agent. The method is more beneficial to the free radical polymerization reaction.

The initiator can be selected from the initiators commonly used in free radical polymerization, preferably Ammonium Persulfate (APS).

The cross-linking agent can be selected from cross-linking agents commonly used in free radical polymerization, preferably N, N' -Methylene Bisacrylamide (MBA); the structural formula of the N, N' -methylene bisacrylamide is shown as a formula V:

the stabilizer can be selected from the stabilizers commonly used in free radical polymerization, preferably Tetramethylethylenediamine (TEMED).

In the step (2), the reaction conditions of the radical polymerization reaction include: the reaction is stirred for at least 2 hours in the solution state, so that the reaction is facilitated. Preferably, the reaction is stirred in a phosphoric acid buffer solution for at least 2 h. In order to achieve a better effect of the invention, it is further preferable that: dissolving an alkene monomer and a cross-linking agent in a Phosphate Buffer Solution (PBS) to obtain a first solution; dissolving an initiator in water to obtain a second solution; and (3) adding the first solution, the second solution and a stabilizer into the reaction solution obtained in the step (1), and stirring for reaction for at least 2 hours. The reaction temperature of the radical polymerization is not strictly limited, and the reaction temperature is generally natural environment temperature, such as normal temperature or room temperature, and the like, and the natural environment temperature is 18-35 ℃.

In the step (2), the mass ratio of the vinyl monomer to the HRP is 20:1, and the mass ratio of the vinyl monomer to the cross-linking agent is 1: 0-1: 0.3; when the mass ratio is 1:0, this is the case where no crosslinking agent is added. The amounts of the initiator and stabilizer are not critical and may be added in the amounts conventionally used for radical polymerization.

In step (2), after the radical polymerization reaction is completed, a purification method commonly used in the art is adopted, such as: and (4) performing ultrafiltration, centrifugation and washing to obtain the purified product nano-gel enzyme.

The raw materials of the invention are all commercial products.

The nano-gelase is prepared according to the preparation method of the nano-gelase. The nano-gel enzyme contains a large amount of zwitterion functional groups, has super-hydrophilicity, has a specific structure capable of maintaining the spatial structure of HRP, improves the environmental stability, effectively resists the adsorptive pollution of degradation products to the HRP, and has wide application prospect in the treatment of industrial wastewater, particularly dye industrial wastewater.

The nanogel enzyme can be directly used as a wastewater treatment material (such as a dye industrial wastewater treatment material), and can also be used as an effective component to be used together with the existing auxiliary materials or auxiliary materials to prepare the wastewater treatment material (such as the dye industrial wastewater treatment material).

The nanogel enzyme can be used for treating industrial wastewater of all dyes (such as basic red dye, indigo carmine dye and other dyes) which can be treated by horseradish peroxidase, and the degradation effect of the nanogel enzyme is obviously better than that of HRP.

The invention has the following advantages:

(1) the preparation method is characterized in that HRP and alkene monomers with zwitterion characteristics are used as raw materials to synthesize the super-hydrophilic nano-gel enzyme, the enzyme is gelated and coated on a molecular level on the premise of not influencing enzyme solubility, the stability of the enzyme is greatly improved, the environmental adaptability of the enzyme is improved, the adsorption of degradation products can be effectively resisted, the recycling and reutilization are facilitated, and the purpose of efficiently and environmentally degrading dyes in industrial wastewater is achieved by utilizing the oxidation capacity of the enzyme;

moreover, the nano gel enzyme material of the horseradish peroxidase is green and has no biological toxicity, and secondary pollution to the environment can not be caused.

(2) The HRP nano-gel enzyme has the advantages of simple synthesis steps, high feasibility, short period, green and environment-friendly synthesis process, mild reaction conditions, low cost and easy industrialization. The method of the invention can greatly improve the thermal stability of the natural enzyme HRP and the degradation performance of the natural enzyme HRP to dye by chemically modifying and synthesizing the gel enzyme.

(3) The nano-gel enzyme has good water solubility, hydrophilicity and biocompatibility, can effectively remove dye, and can be directly used as a degradation material of dye industrial wastewater. For the same dye, both HRP and the nano-gel enzyme have degradation effect on the dye, but the degradation effect of the nano-gel enzyme is far higher than that of the HRP; under the same degradation time, the degradation effect of the nanogel enzyme on the dye is far better than that of HRP, and the effect is obvious.

Drawings

FIG. 1 is a graph of concentration versus absorbance for various materials at different concentrations in example 2; wherein the ordinate is the absorbance (OD) at wavelength 335nm₃₃₅) The abscissa represents the Concentration (. mu.g/mL).

FIG. 2 is a graph showing the activity detection effect of nanogel enzyme and HRP at the same concentration in example 3 at 55 ℃; wherein the ordinate is the absorbance (OD) at a wavelength of 450nm₄₅₀) The abscissa is time, minutes (min).

FIG. 3 is a graph showing the effect of detecting the activity of nanogel enzyme and HRP at different concentrations for 20min at a constant temperature of 55 ℃ in example 4; wherein the ordinate is the absorbance (OD) at a wavelength of 450nm₄₅₀) The abscissa represents the Concentration (. mu.g/mL).

FIG. 4 is a graph showing the effect of nanogel enzyme and HRP on the degradation of basic red dye in example 7; wherein the ordinate is the absorbance (OD) at 545nm₅₄₅) The abscissa is time, minutes (min).

FIG. 5 is a graph showing the effect of nanogel enzyme and HRP on the removal rate of basic red dye at different pH values in example 7, wherein the ordinate is percent removal rate (percent) and the abscissa is pH value.

FIG. 6 is a graph showing the effect of nanogel enzyme (CBAA monomer) and HRP on the degradation of indigo carmine dye in example 8; wherein the ordinate is the absorbance (OD) at a wavelength of 610nm₆₁₀) The abscissa is time, minutes (min).

FIG. 7 is a graph showing a particle size distribution of the nanogel enzyme synthesized in example 1; wherein the ordinate is Intensity (%); the abscissa is the radius (radius), nm.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and examples, but the present invention is not limited thereto.

Example 1

(1) Preparation of HRP-NAS monomer:

the molecular weight of the HRP is 44000 Da;

weighing HRP5.0mg, placing in a micro reaction bottle, weighing 2mL of 0.2mol/L PBS, adding into the micro reaction bottle, and stirring at room temperature to dissolve HRP; dissolving 5.0mg of NAS in 0.25mL of DMSO to obtain a NAS solution; slowly dripping the NAS solution with the molar quantity of the NAS to the molar quantity of the amino groups in the HRP being 6:1 into a micro reaction flask, stirring at room temperature, and reacting for 2 hours to obtain the HRP-NAS solution.

(2) Preparation of HRP nanogel enzyme (GEL-1):

dissolving 100mg of CBAA and 20mg of MBA in 2.5mL of 0.2mol/L PBS to obtain a first solution; dissolving 4mg APS in 0.5mL deionized water to obtain a second solution; and (3) completely adding the first solution and the second solution and 15 mu L of TEMED into the HRP-NAS solution obtained in the step (1), stirring at room temperature for reacting for 2 hours, completely transferring the reaction solution into an ultrafiltration centrifugal tube, centrifuging and washing for 6 times to obtain a product, namely the HRP nanogel enzyme (GEL-1). The product is nano-scale, and the detection result of Dynamic Light Scattering (DLS) is shown in figure 7.

Example 2

Determination of the number of double bonds in HRP-NAS monomer.

The solution of glycine (Gly), HRP and HRP-NAS prepared in example 1 is diluted by gradient with 0.01mol/L PBS solution; diluting 2,4, 6-trinitrobenzenesulfonic acid (TNBS) by 100 times with 0.1mol/L aqueous solution of sodium bicarbonate to obtain TNBS solution, and simultaneously preparing 10 mass percent aqueous solution of Sodium Dodecyl Sulfate (SDS) and 1mol/L aqueous solution of HCl.

In a 96-well plate, 120. mu.L of Gly, HRP and HRP-NAS solution with each concentration diluted by gradient is respectively added and marked, then 60. mu.L of TNBS solution is added into each well, the mixture is cultured at the constant temperature of 37 ℃ for 2 hours, then 60. mu.L of SDS aqueous solution and 30. mu.L of HCl aqueous solution are added, the reaction is stopped, and the OD value is measured by using a microplate reader.

The structural formula of glycine is shown in formula VI:

the structural formula of the 2,4, 6-trinitrobenzenesulfonic acid (TNBS) is shown as a formula VII:

the degree of grafting of the amino groups is determined by the slope of the straight line. The slopes of HRP and HRP-NAS were gradually decreased by the comparison with glycine as a reference, as shown in FIG. 1. It was confirmed that the target compound HRP-NAS was synthesized by the reaction, and on average 9.71 double bonds were grafted per HRP.

The number of grafted amino groups (equivalent to the number of double bonds in the HRP-NAS monomer) can be calculated from a glycine standard curve using glycine solution per unit concentration. The number of amino acids on the protein surface is then:

the number of amino groups reacted on the HRP surface was 12.18, and the number of amino groups remaining after grafting was 2.47, indicating that the number of grafted amino groups was 9.71.

TABLE 1 degree of grafting of HRP surface amino groups to NAS

	Gly	HRP	HRP-NAS
				Slope	0.04	8.31×10^-4	1.69×10^-4
A	1	48.13	236.68
				Mw/(75*A)	1	12.18	2.47

Note: slope represents the rate at which TNBS is quantified by the amino number consumption of the sample solution;

a is expressed as the ratio of the rates at which one amino group is consumed [ Slope/Slope (Gly) ].

Example 3

Detection of catalytic Activity of Nanogel enzyme (GEL-1) prepared in example 1.

After the nanogel enzyme (GEL-1) and HRP were allowed to stand at the same concentration at a constant temperature of 55 ℃ for various periods of time, the activity thereof was examined as follows.

50 mu.L of nanogel enzyme (GEL-1) and HRP with the same concentration are added into a 96-well plate and labeled (GEL-1 and HRP are diluted by 0.01mol/L PBS solution), then 100 mu.L of Tetramethylbenzidine (TMB) is added into each well, after 5min, 50 mu.L of 1mol/L HCl aqueous solution is added to stop the reaction, then the OD value is measured by a microplate reader, and the detection result is shown in figure 2.

The structural formula of the tetramethylbenzidine is shown as formula VIII:

example 4

Environmental suitability assay of nanogel enzyme (GEL-1) prepared in example 1.

HRP and nanogel (GEL-1) with different concentrations were incubated at 55 ℃ for a period of time, and then activities were measured using TMB and a microplate reader according to the ratio and method for measuring activities in example 3. The results are shown in FIG. 3.

Under the same concentration, the thermal stability of the nanogel enzyme is far higher than that of HRP, and the nanogel enzyme still keeps certain activity at 180min, as shown in figure 2; for nanogel enzymes with different concentrations and HRP at the constant temperature of 55 ℃ for the same time, the thermal stability of the nanogel enzyme is also higher than that of the HRP, and the higher the concentration is, the better the thermal stability is, and a certain activity is also kept at 10ng/mL, as shown in FIG. 3; therefore, the heat stability of the natural enzyme HRP can be greatly improved by chemically modifying HRP according to the method for synthesizing the gel enzyme.

Example 5

(1) Preparation of HRP-NAS monomer:

the molecular weight of the HRP is 44000 Da;

weighing HRP5.0mg, placing in a micro reaction bottle, weighing 2mL of 0.2mol/L PBS, adding into the micro reaction bottle, and stirring at room temperature to dissolve HRP; dissolving 5.0mg of NAS in 0.25mL of DMSO to obtain a NAS solution; slowly dripping the NAS solution with the molar quantity of the NAS to the molar quantity of the amino groups in the HRP being 8:1 into a micro reaction flask, stirring at room temperature, and reacting for 2 hours to obtain the HRP-NAS solution.

(2) Preparation of HRP nanogel enzyme (GEL-2):

dissolving 100mg of SBMA and 20mg of MBA in 2.5mL of 0.2mol/L PBS to obtain a first solution; dissolving 4mg APS in 0.5mL deionized water to obtain a second solution; and (3) completely adding the first solution and the second solution and 15 mu L of TEMED into the HRP-NAS solution obtained in the step (1), stirring at room temperature for 2h, completely transferring the reaction solution into an ultrafiltration centrifugal tube, centrifuging and washing for 6 times to obtain a product, namely the HRP nanogel enzyme (GEL-3). The product is nano-scale, and the particle size is 67-72 nm.

The method for determining the number of double bonds in the HRP-NAS monomer prepared in this example is the same as that of example 2. The slopes of HRP and HRP-NAS were gradually decreased by comparison with glycine as a reference, demonstrating that the target compound HRP-NAS was synthesized by the reaction, with an average of 2.89 double bonds grafted per HRP.

The method for detecting the catalytic activity of nanogel enzyme (GEL-2) prepared in this example is the same as that of example 3. The environmental suitability test method of nanogel enzyme (GEL-2) prepared in this example is the same as that of example 4. And (3) displaying a detection result: under the same concentration, the thermal stability of the nanogel enzyme is far higher than that of HRP, and certain activity is still kept at 180 min; for the nanogel enzyme with different concentrations and the HRP, the thermal stability of the nanogel enzyme is also higher than that of the HRP at the constant temperature of 55 ℃ for the same time, and the higher the concentration is, the better the thermal stability is, and a certain activity is also kept at 10 ng/mL; therefore, the heat stability of the natural enzyme HRP can be greatly improved by chemically modifying HRP according to the method for synthesizing the gel enzyme.

Example 6

(1) Preparation of HRP-NAS monomer:

the molecular weight of the HRP is 44000 Da;

weighing HRP5.0mg, placing in a micro reaction bottle, weighing 2mL of 0.2mol/L PBS, adding into the micro reaction bottle, and stirring at room temperature to dissolve HRP; dissolving 5.0mg of NAS in 0.25mL of DMSO to obtain a NAS solution; slowly dripping the NAS solution into a micro reaction bottle according to the ratio of the molar quantity of the NAS to the molar quantity of the amino group in the HRP (horse radish peroxidase) of 3:1, stirring at room temperature, and reacting for 2 hours to obtain an HRP-NAS solution.

(2) Preparation of HRP nanogel enzyme (GEL-3):

dissolving 100mg of MPC and 20mg of MBA in 2.5mL of 0.2mol/L PBS to obtain a first solution; dissolving 4mg APS in 0.5mL deionized water to obtain a second solution; and (3) completely adding the first solution and the second solution and 15 mu L of TEMED into the HRP-NAS solution obtained in the step (1), stirring at room temperature for 2h, completely transferring the reaction solution into an ultrafiltration centrifugal tube, centrifuging and washing for 6 times to obtain a product, namely the HRP nanogel enzyme (GEL-3). The particle size of the product was measured using DLS and ranged from 78 nm to 80 nm.

The method for determining the number of double bonds in the HRP-NAS monomer prepared in this example is the same as that of example 2. The slopes of HRP and HRP-NAS were gradually decreased by comparison with glycine as a reference, demonstrating that the target compound HRP-NAS was synthesized by the reaction, with an average of 3.6 double bonds grafted per HRP.

The method for detecting the catalytic activity of nanogel enzyme (GEL-3) prepared in this example is the same as that of example 3. The environmental suitability test method of nanogel enzyme (GEL-3) prepared in this example is the same as that of example 4. And (3) displaying a detection result: under the same concentration, the thermal stability of the nanogel enzyme is far higher than that of HRP, and certain activity is still kept at 180 min; for the nanogel enzyme with different concentrations and the HRP, the thermal stability of the nanogel enzyme is also higher than that of the HRP at the constant temperature of 55 ℃ for the same time, and the higher the concentration is, the better the thermal stability is, and a certain activity is also kept at 10 ng/mL; therefore, the heat stability of the natural enzyme HRP can be greatly improved by chemically modifying HRP according to the method for synthesizing the gel enzyme.

Example 7

Degradation of basic red (BR29) dye by nanogel enzyme (GEL-1) prepared in example 1:

two 50ppm aqueous solutions of BR29 dye were prepared, 10mM (mmol/L) aqueous hydrogen peroxide was added to adjust the pH to 5.64, and a stirring speed of 500rpm was maintained at 40 ℃ C. in which HRP was added in one portion and nanogel (GEL-1) was added in the other portion, and the total volume of each portion was 10mL, and the concentration of HRP and nanogel in each portion was 50. mu.g/mL.

The structural formula of the basic red dye is shown as formula IX:

over time, HRP can degrade basic red dye to one-half of the original, while nanogel enzyme of the invention can degrade basic red dye almost completely, as shown in fig. 4; therefore, for the same dye, both HRP and the nano-gel enzyme have degradation effect, but the degradation effect of the nano-gel enzyme is far higher than that of the HRP; under the same degradation time, the nanogel enzyme has a remarkable degradation effect on basic red dye. The degradation effects of nanogel enzyme and HRP under different pH values are shown in FIG. 5; the degradation effect of the basic red dye under the alkaline condition is better than that of the basic red dye under the acidic condition, and the degradation effect of the nano-gel enzyme under the acidic condition is better than that of the HRP (horse radish peroxidase), which shows that the nano-gel enzyme has better degradation effect on the dye in a wider pH value range.

Degradation experiments of basic red (BR29) dye by nanogel enzyme (GEL-2) prepared in example 5 and nanogel enzyme (GEL-3) prepared in example 6 were the same as those of basic red (BR29) dye by nanogel enzyme (GEL-1) prepared in example 1. The experimental results show that: with the lapse of time, HRP can degrade basic red dye to half of the original, and the nanogel enzyme of the invention can degrade basic red dye almost completely; therefore, for the same dye, both HRP and the nano-gel enzyme have degradation effect, but the degradation effect of the nano-gel enzyme is far higher than that of the HRP; under the same degradation time, the nanogel enzyme has a remarkable degradation effect on basic red dye. Under different pH values, the degradation effect of the basic red dye under the alkaline condition is better than that of the basic red dye under the acidic condition, and the degradation effect of the nano-gel enzyme under the acidic condition is better than that of HRP (horse radish peroxidase), which indicates that the nano-gel enzyme has better degradation effect on the dye in a wider pH value range.

Example 8

Degradation of indigo carmine dye by nanogel enzyme (GEL-1) prepared in example 1.

Two 100ppm aqueous solutions of indigo carmine dye were prepared, 10mM aqueous hydrogen peroxide was added to each solution, the pH was adjusted to 5.93, and a stirring speed of 500rpm was maintained at 40 ℃ while HRP was added to one of the solutions and nanogel (GEL-1) was added to the other, the total volume of each solution was 10mL, and the concentration of HRP and nanogel (GEL-1) in each solution was 50. mu.g/mL.

The structure of indigo carmine is shown in formula X:

over time, HRP can degrade the indigo carmine dye to half of the original, while the nanogel enzyme of the invention can degrade the indigo carmine dye almost completely, as shown in fig. 6; therefore, for the same dye, both HRP and the nanogel enzyme have the degradation effect, but the degradation effect of the nanogel enzyme is far higher than that of the HRP.

Example 9

Degradation of indigo carmine dye by nanogel enzyme (GEL-2) prepared in example 5 and nanogel enzyme (GEL-3) prepared in example 6.

Two 100ppm aqueous solutions of indigo carmine dye were prepared, 10mM aqueous hydrogen peroxide was added to each solution, the pH was adjusted to 5.93, and a stirring speed of 500rpm was maintained at 40 ℃ while HRP was added to one of the solutions and nanogel enzyme (GEL-2) or nanogel enzyme (GEL-3) was added to the other, the total volume of each solution was 10mL, and the concentration of HRP and nanogel enzyme in each solution was 50. mu.g/mL.

Over time, the HRP can degrade the indigo carmine dye to one half of the original, while the nanogel enzyme of the invention can almost completely degrade the indigo carmine dye; it can be seen that for the same dye, both HRP and the nanogel enzyme of the invention have the degradation effect, but the degradation effect of the nanogel enzyme of the invention is far higher than that of the HRP.

The change of the parameters in the preparation method does not influence the preparation of the nano-gel enzyme, so that the preparation of the nano-gel enzyme can be realized by the combination of any parameter in the preparation method. And will not be described in detail herein.

Claims

1. A preparation method of nanogel enzyme is characterized by comprising the following steps:

(1) chemically modifying horseradish peroxidase by using N-acryloyloxy succinimide to prepare a horseradish peroxidase-N-acryloyloxy succinimide macromonomer;

(2) carrying out free radical polymerization reaction on the horseradish peroxidase-N-acryloyloxy succinimide macromonomer obtained in the step (1) and a vinyl monomer with zwitterion characteristic to prepare nanogel enzyme;

the vinyl monomer with zwitterion characteristics is one or more than two of carboxylic acid betaine acrylamide, carboxylic acid betaine methacrylate, sulfobetaine methacrylate and 2-hydroxyethyl methacrylate phosphatidylcholine;

in the step (2), three raw materials of an initiator, a stabilizer and a cross-linking agent are also added in the free radical polymerization reaction; the cross-linking agent is N, N' -methylene bisacrylamide.

2. The method for preparing nanogel enzyme according to claim 1, wherein in the step (1), the reaction conditions for chemically modifying horseradish peroxidase by using N-acryloyloxy succinimide comprise: stirring and reacting for at least 2h in a solution state;

in the step (2), the reaction conditions of the radical polymerization reaction include: the reaction was stirred in solution for at least 2 h.

3. The method for preparing nanogel enzyme according to claim 2, wherein in the step (1), the reaction conditions comprise: dissolving horseradish peroxidase in a phosphoric acid buffer solution to obtain a horseradish peroxidase solution; dissolving N-acryloyloxy succinimide in dimethyl sulfoxide to obtain an N-acryloyloxy succinimide solution; stirring a horseradish peroxidase solution, dropwise adding an N-acryloyloxy succinimide solution into the horseradish peroxidase solution, and stirring for reaction for at least 2 hours;

in the step (2), the reaction conditions of the radical polymerization reaction include: the reaction was stirred in phosphoric acid buffer solution for at least 2 h.

4. The method for preparing nanogel enzyme according to claim 1, wherein in the step (1), the N-acryloyloxy succinimide is used in an amount that: the molar amount of N-acryloyloxy succinimide is greater than or equal to the molar amount of amino groups in horseradish peroxidase.

5. A nanogel enzyme, which is produced by the method for producing a nanogel enzyme according to any one of claims 1 to 4.

6. The use of nanogel enzyme according to claim 5 directly as a dye industrial wastewater treatment material or for the preparation of a dye industrial wastewater treatment material.